The seeds of land plants contain large quantities of storage, or reserve proteins which are synthesized during the development of the seeds. During germination and early seedling growth, these reserves are hydrolyzed to produce metabolic intermediates for use by the growing seedling. In harvested seeds, storage proteins represent an available package of condensed food and enzymes. The food value of these seeds would be increased by altering the composition of the reserve proteins to decrease the amount of undesirable proteins in the seeds.
Some of the seed storage proteins in most, if not all, plants are in a class called protease inhibitors. These inhibitors are thought to function not only as storage proteins, but as regulators of endogenous proteases, and as proteins that protect plants from insect and pathogen attack.
The plant protease inhibitors are generally low molecular weight proteins, and share in common the ability to combine with particular animal, and occasionally plant proteases, thereby abolishing the activity of these enzymes. The literature suggests that active protease inhibitors may be toxic to humans and other animals, adversely affecting the nutritional quality of plant foodstuffs. Thus, there is a need to minimize the amount of protease inhibitors in foods.
Protease inhibitors are particularly abundant in the legume family and constitute about 6% of the proteins of soybeans. See Brandon, U.S. Pat. No. 4,959,310, incorporated herein by reference. Their antinutritional nature has been linked to pancreatic hyperplasia, acinar adenoma, and overall growth reduction when raw soybean meal is fed to monogastric animals, such as chickens, rats, and quail.
Soybean (Glycine max) seed proteins are one example of storage proteins that are widely used in human foods such as infant formulas, tofu, soy protein isolates, soy flour, textured soy fibers and soy sauce. Soybean protein products serve as an excellent source of low cost, high quality protein for human needs. Soybeans are also widely used as a component of animal feeds. However, they must undergo costly processing to properly remove or deactivate protease inhibitors.
Soybean protease inhibitors are categorized into three classes: Kunitz trypsin inhibitors ("KTI"), Bowman-Birk inhibitors ("BBI"), and glycine-rich soybean trypsin inhibitors ("GRSTI"). The primary structure of these inhibitors consists partly of sulfur-containing (methionine and cysteine) amino acids. Killipara, K. P. and Hymowitz, T., J. Agr. Food, Vol. 40, pp. 2356-2363, (1992), incorporated herein by reference.
The major, predominantly expressed form of KTI's is a 21.5-KDa protein which has an inhibition specificity for trypsin. BBI is a low molecular weight (8000 kDa) protein that inhibits both trypsin and chymotrypsin simultaneously at independent reactive sites. At least ten different isoforms of BBI have been reported. GRSTI's are minor inhibitors of trypsin in soybean seed.
Various approaches have been taken to reduce the protease inhibitor content and/or activity of soybeans. These include physical (heat) and chemical treatment of soy products, as well as the genetic alteration of soybeans through conventional breeding techniques.
In any heat treatment, care must be taken because, even though heating is required to destroy the trypsin inhibitors, improper heating will result in damage to the protein product itself. Furthermore, although the protease inhibitor activity is largely inactivated by denaturation through conventionally applied heat treatment of soy flour, 10-15% residual activity usually remains. The unusual structure of BBI is the most likely reason for this residual activity. BBI is strongly cross-linked by disulfide bonds which gives the molecule resistance to heat denaturation. Thus, heat treatment of seed or soy products to reduce inhibitor expression is not completely successful and furthermore, involves costly energy usage.
The solvent-extraction method is another process used to eliminate protease inhibitors from raw soybeans. This chemical extraction, while removing the various inhibiting materials, results in considerable loss of the oil in the seed, thus reducing its food value. At the same time, the solvent poses problems of cleanup and disposal.
Genetic modification of the soybean plant to develop low inhibitor activity varieties has also been proposed, but has inherent limitations. Desirable nutritional value may be lost concomitant with reduction of the inhibitors, and cross pollination of the genetic variant with another cultivar could result in reexpression of the protease inhibitor gene. Further, altering expression of one inhibitor may not affect the expression of another.
As yet, conventional breeding and tissue culture technology have been unable to produce a soybean plant,with low levels of protease inhibitors, although a need exists for such plants. Breeders have attempted to use genes lacking KTI due to mutations of the gene. See e.g. Zhang, et al., "Effects of Extrusion and Expelling on the Nutritional Quality of Conventional and Kunitz Trypsin Inhibitor-Free Soybeans"; Poultry Science, Vol. 72; pp. 2299-2308; (1993); incorporated herein in its entirety by reference. It is known that KTI-free soybean has a genetic difference which results in a 40 to 50% decrease in trypsin inhibitor activity. See e.g. Friedman, et al., J. Agric. Food Chem.; Vol. 39; pp. 327-335; (1991); and Anderson-Hafermann, et al., Poultry Sci.; Vol. 71; pp. 1700-1709; (1992); both incorporated herein in their entirety by reference. However, although the aforementioned reductions in levels of protease inhibitors ("PI") is unprecedented and significant, the levels are not low enough to completely eliminate the need to inactivate the remaining PI for animal feed. Since the PI in soybean, for example, are contributed from three different classes of inhibitors and each of these classes is comprised of proteins coded by multiple genes, there are no known means to genetically alter all classes of PI with a common method.
Until now, only one method has been illustrated to result in the reduction of all protease inhibitors in the plant seeds. The method referred to is the introduction of a foreign gene from Brazil nut encoding higher levels of methionine-rich 2S seed storage protein such as Brazil nut protein ("BNP"). Altenbach, et al., Plant Mol. Biol., Vol. 8; pp. 239-250; (1987); incorporated herein in its entirety by reference. The introduction of the BNP gene resulted in a 60% reduction in overall trypsin inhibitor activity and a 90% reduction in overall .alpha.-chymotrypsin inhibitor activity. However, the reduction in levels of protease inhibitors is still not sufficient.
Based on the foregoing, there is a need to provide plant seeds and feed products that would not need processing before feeding to monogastric animals and/or humans.
It is therefore an object of the present invention to provide a novel method to eliminate or reduce the content of endogenous proteins in plant seeds.
It is a further object of the present invention to provide plant seeds that do not require time consuming or costly processing to eliminate protease inhibitor activity.
It is a further object of this invention to provide a transgenic plant that produces a seed having little or no protease inhibitor content.